Humans lack the capacity to synthesize omega-3 polyunsaturated fatty acids (n-3 PUFAs); thus, these essential nutrients must be obtained from the diet. Pelagic fish are a rich source of the long chain n-3 PUFAs, docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA), while oil derived from plants such as flaxseed and soybean are excellent sources of the shorter chain n-3 PUFA, α-linolenic acid (ALA). Although national and international health and government organizations provide recommendations for dietary intake of n-3 PUFAs, targets are rarely met by most populations. Furthermore, segments within these populations may have particularly low intake and/or bioavailability of n-3 PUFAs that correlate with actual or increased risk of disease. Specific examples include babies that are fed nonfortified infant milk formula, individuals with polymorphic genes encoding enzymes with low efficacy for transport of fatty acids across membranes, and vegan populations with negligible EPA and DHA intake. The relationship between n-3 PUFA intake and n-3 PUFA bioavailability is complex and influenced by many factors, including genetics, smoking status, age, gender, and ratio of n-6 to n-3 PUFAs in the diet of the individual. The omega-3 index is a measure of the combined EPA plus DHA content of red blood cell membranes, expressed as a percentage of total membrane fatty acids, and serves as a better indicator of an individual’s n-3 PUFA status than measurements of dietary intake. This chapter examines evidence highlighting associations between n-3 PUFA deficiency and adverse impacts on human mental and physical health. While the focus is on human n-3 PUFA deficiency, findings from cell and animal-based research have been included where this contributes toward improved understanding of human deficiency. The chapter also explores new and developing methods for production of n-3 PUFAs that promise clean and sustainable alternatives to fish, to address n-3 PUFA deficiency in human populations.